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Effect of smoking on serum xanthine oxidase and malondialdehyde levels in patients with acute myocardial infarction.

Byline: Muhammad Atif Ata Fatehuddin Khand Shumaila Shabbir Shaikh and Muhammad Adeel Ata

Abstract

Objective: To evaluate the effect of smoking on blood levels of xanthine oxidase and malondialdehyde in acute myocardial infarction patients.

Methods: The cross-sectional study was conducted from June to November 2013 and comprised myocardial infarction in-patients at Liaquat University of Medical and Health Sciences Hospital Jamshoro and Isra University Hospital Hyderabad. Serum samples from age and gender matched smoking and non-smoking patients were investigated for the levels of xanthine oxidase and malondialdehyde by kit methods.

Results: Of the 88 serum samples investigated 68(77.3%) belonged to men and 20(22.7%) to women. Among the 44 smokers 35(79.5%) were men and 9(20.5%) were women. The overall mean age of the group was 49.68.0 years. Among the 44 non-smokers 33(75%) were men and 11(25%) women. The overall mean age of the group was 50.98.4 Years. Smokers compared to non-smokers had significantly raised mean serum xanthine oxidase (0.310.05 vs 0.280.03mg/dl; pless than 0.05) and malondialdehyde (32.293.30 vs 30.302.87 mol/L; pless than 0.04) levels.

Conclusion: Smokers as against the non-smokers were at higher risk of developing acute myocardial infarction owing to increased level of oxidative stress caused by smoking.

Keywords: Xanthine oxidase Malondialdehyde Myocardial infarction Smoking. (JPMA 65: 39; 2015)

Introduction

The effects of smoking on human health are detrimental and in many cases lethal. Cigarette smoke carries approximately 4000 chemicals including numerous toxic metals poisonous gases and free radicals.1 These ingredients in smoke when inhaled regularly can cause oxidative stress (OS) via biotransformation or by macrophage activation.2 Smoking is now recognised as a major risk factor of acute myocardial infarction (AMI) especially in men.3 AMI is the leading cause of death in the developed countries.4 The prevalence of AMI increases progressively with age and peaks between the ages 45 and 65 years. Men are four to five times more likely to develop MI than women.4

Different biomarkers have been identified for the detection of myocardial damage. These include aspartate aminotransferase (AST) lactate dehydrogenase (LDH) creatine kinase (CK) and troponins I and T. In the assessment of myocardial injury these markers are of not much value because of certain limitations.5 In human beings xanthine oxidase (XO) is normally present in liver and in the myocardium but is absent in the blood. During MI XO is released from the myocardium into the blood so a blood assay for XO can be used to assess AMI patients. XO is important as it has been shown to be a major source of free radicals generation under ischaemic conditions.67

Malondialdehyde (MDA) is one of the final products of lipid peroxidation by reactive oxygen species (ROS) generated in cells.8 The production of MDA is used as a biomarker to measure OS level in an organism.910 In the current study serum XO and MDA levels were measured in smoking and non-smoking AMI patients in order to see the effect of smoking on the levels of these blood parameters.

Patients and Methods

The cross-sectional study was conducted from June to November 2013 and comprised myocardial infarction in- patients at Liaquat University of Medical and Health Sciences Hospital Jamshoro and Isra University Hospital Hyderabad. Initially 115 MI patients were interviewed regarding their smoking habits. The responses thus obtained were recorded on a pre-designed proforma. Informed written consent was obtained from all patients and the approval was granted by the Ethical Committee of Isra University. From among the respondents two age and gender matched groups of smokers and non-smokers were evaluated for serum XO and MDA levels which were measured by Bio Vision kit and oxiselect TM MDA Adduct enzyme-linked immunosorbent assay (ELISA) kit respectively.

XO oxidises xanthine to hydrogen peroxide (H2O2) which reacts stoichiometrically with OxiRedTM Probe to generate colour which is measured at 570nm. As for the reagents OxiRedTM Probe Preparation was prepared by dissolving the probe in 220l of dimethylsulfoxide (DMSO); XO Enzyme Mix was prepared by dissolving XO enzyme mixture in 220l deionised water; XO Substrate Mix was prepared by dissolving XO substrate mixture in 220l deionised water; and XO Positive Control was prepared by diluting XO positive control with 92l deionised water.

XO assay protocol was followed and 10mM H2O2 standard was prepared by diluting 4 l 0.88mM H2O2 standard with 348l deionised water; 0.1mM H2O2 standard was prepared by diluting 10 l of 10mM H2O2 standard with 990 l deionised water. For the colorimetric assay 0 10 20 30 40 and 50l of 0.1mM H2O2 standard were transferred into 96 well plate in duplicates and deionised water was added to make the final volume to 50l in each well. Thus working standards of 0 1 2 3 4 and 5nM H2O2 were generated.

For sample and positive control preparations the serum was directly added to sample wells and the final volume was adjusted to 50l/well with deionised water. Positive control was prepared by putting 5l control solution to wells and adjusting the volume to 50l/ well with deionised water.

After adding 50 l/well reaction mix in H2O2 standards test samples and positive controls the contents in wells were mixed and immediately read for absorbance at 570nm. Results were determined via calibration curve.

For the measurement of MDA by OxiSelectTM MDA Adduct ELISA kit Bovine serum albumin (BSA) standards and protein samples (10g/ml) were adsorbed onto a 96-wellplate for 2 hours at 37C. The MDA-protein adducts present in the sample or standard were probed with an anti-MDA antibody followed by a horseradish peroxidase (HRP) conjugated secondary antibody. The MDA protein adducts content in an unknown sample was determined by comparing with a standard curve that was prepared from pre-determined MDA-BSA standards.

For reagents preparation 1X wash buffer was prepared by diluting 10X wash buffer concentrate to 1X with deionised water and stirred to homogeneity; anti-MDA antibody and secondary antibody was diluted 1:1000 each with assay diluent and was used freshly.

The 10g/ml standard curve of reduced BSA was freshly prepared by diluting the 1mg/ml BSA standard in 1X phosphate buffered saline (PBS) and the 0.5g/ml of MDA-BSA standard curve was freshly prepared by diluting the 1mg/ml MDA-BSA standard in 10 g/ml of reduced BSA.

The MDA assay protocol was followed. Each serum sample and MDA-BSA standard were assayed in duplicate.

In the 1st incubation 100l of the serum samples or reduced/MDA-BSA standards were put in 96 well protein- binding plates and incubated at 37C for 3 hours. The wells were then washed twice with 250l 1X PBS per well. After the last wash wells were emptied and tapped to remove excess wash solution.

In the 2nd incubation 200l of assay diluents/well were added and incubated for 1-2 hours at room temperature on an orbital shaker. The wells were then washed thrice with 250l of 1X wash buffer with thorough aspiration between each wash. After the last wash the wells were emptied to remove excess 1X wash buffer.

In the 3rd incubation 100L of the diluted anti-MDA antibody was added to all the wells and incubated for 1hr at room temperature on an orbital shaker. This was followed by washing step as in the 2nd incubation.

In the 4th incubation 100L of the diluted secondary antibody-HRP conjugate was added to all the wells and incubated for 1hr at room temperature on an orbital shaker.

Additional 100l of warmed substrate solution was added to each well including the blank well and the contents were incubated at room temperature on an orbital shaker. The enzyme reaction was stopped by adding 100L of stop solution to each well. Absorbance of each well was then immediately measured on a microplate reader using 450nm as the primary wavelength and reduced BSA standard as the absorbance blank.

All data was presented as mean standard deviation (SD). Comparison of mean values between smokers and non- smokers with MI were made by using unpaired student t test. Differences between the two groups were considered significant at pless than 0.05.

Results

Of the 115 AMI patients initially approached 60(52.2%) were smokers and 55(47.8%) were non-smokers. Of the 88(76.5%) subjects who formed the main study sample 68(77.3%) were men and 20(22.7%) were women. Among the 44 smokers 35(79.5%) were men and 9(20.5%) were women. Men irrespective of whether they were smokers or non-smokers were more than three times likely to have

Table-1: Gender distribution.

MI patient Group###Gender###Male to Female

###Male###Female###ratio

Smokers (n=44)###35 (79.5%)###09 (20.5%)###3.9:1

Non-smokers (n=44)###33 (75%)###11 (25%)###3:1

Total###68###20###3.4:1

Table-2: Comparison between smokers and non-smokers.

Serum###Smokers with###Non-smokers with###P Value Significance

Variable###AMI ( n=44)###AMI ( n=44)###level

###MeanS.D.###MeanS.D.

XO (mg/dl)###0.3080.05###0.2810.027###P=0.05###P = 0.05

MDA (mol/L)###32.293.30###30.302.87###P=0.04###is significant

AMI compared to women (Table-1).

The overall mean age of the group was 49.68.0 years. Among the 44 non-smokers 33(75%) were men and 11(25%) women. The overall mean age of the group was 50.98.4years.

Smokers compared to non-smokers had significantly raised mean serum xanthine oxidase (0.310.05 vs 0.280.03mg/dl; pless than 0.05) and malondialdehyde (32.293.30 vs 30.302.87 mol/L; pless than 0.04) levels (Table 2).

Discussion

AMI is the predominant cause of death in both developed and developing countries. It is a multi-factorial disease and cigarette smoking is one of the major contributing factors for atherosclerosis and hence of AMI.

The finding of the current study that males are more than three times more likely to develop AMI than the age- matched females is in full agreement with literature11 and supports the notion that males are four to five times more prone to developing MI than females.4

A study12 reported higher levels of XO in MI patients irrespective of their smoking status. XO levels are usually raised in ischaemia reperfusion injuries anoxia and inflammation. In the present study the effect of smoking which is an important source of oxygen free radicals on the serum levels of XO which is a potential source of free radicals generation in ischaemic conditions and MDA in AMI patients were investigated. Both serum XO and MDA which is a marker of OS levels were significantly raised in smokers with AMI than in non-smokers with AMI. This finding suggests that significantly higher levels of XO in smokers with AMI has increased the generation of reactive oxygen species (ROS) which in turn has increased the lipid peroxidation manifested by high levels of MDA.

Our finding that MDA levels are significantly higher in smoking than in non-smoking AMI patients is in line with earlier reports1314 that found that regular smokers had a slightly higher average concentration of MDA than the non-smokers.

Since the concentration of free radicals in smoke is extremely high smoking by inducing superoxide radical production in vascular endothelial cells could cause endothelial dysfunction owing to conversion of nitric oxide into peroxynitrite.15 Smoking-induced OS also results in increased low-density lipoprotein (LDL) oxidation which in turn facilitates the formation of the atheromatous plaque.16 In this way smokers are at higher risk of developing AMI and chronic heart diseases (CHD) than the non-smokers.

Regarding the finding of high levels of XO in serum samples of smokers as against the non-smokers it is not clear at this stage as to why and how the level of XO increases more in smoking AMI patients and this aspect needs to be thoroughly investigated by future researchers.

Conclusion

Smoking AMI patients were at a higher risk of developing OS and hence of AMI than the non-smokers.

Acknowledgment

We are grateful to the management of Isra University for providing financial assistance.

References

1. Schumacher JN Green CR Best FW Newell MP. Smoke composition: an extensive investigation of the water soluble portion of cigarette smoke. J Agric Food Chem 1997; 25: 310-20.

2. Nawrot TM Cox B Janssen BG Plusquin M. Prevention-Passive smoking and pregnancy. EJC Supplements 2013; 11: 242-7.

3. Teo KK Ounpuu S Hawken S Pandey MR Valentin V Hunt D et al. Tobacco use and risk of myocardial infarction in 52 countries in the Inter heart study: a case-control study. Lancet 2006; 368: 647-58.

4. Marrugat J Sala J Aboal J. Epidemiology of cardiovascular disease in women. Rev Esp Cardiol 2006; 59: 264-74.

5. Kemp M Donovan J Higham H Hooper J. Biochemical markers of myocardial injury. Br J Anaesth 2004; 93: 63-73.

6. Gorman SL Zweier JL. Evaluation of the role of xanthine oxidase in myocardial reperfusion injury. J Biol Chem 1990; 265: 6656-63.

7. Bhakuni P Chandra M Misra MK. Oxidative stress parameters in erythrocytes of post reperfused patients with myocardial infarction. J EnzIhibi Med Chem 2005; 20: 377-81.

8. Harrison R. Structure and function of xanthine oxidoreductase: where are we now Free Radic Biol Med 2002; 33: 774-97.

9. Moore K Roberts LJ. Measurement of lipid peroxidation. Free Radic Res 1998; 28: 659 - 71.

10. Del Rio D Stewart AJ Pellegrini N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis 2005; 15: 316-28.

11. Smith WC Kenicer MB Tunstall-Pedoe Clark EC Crombie IK. Prevalence of coronary heart disease in Scotland : Scottish Heart Health Study. Br Heart J 1990; 64: 295-8.

12. Raghuvanshi R Chandra M MISRA PC MISRA MK. Effect of vitamin E on the patients of myocardial infarction. Ind. J Clinical Biochem 2005; 20: 26-9.

13. Nielsen.F Mikkelsen BB Nielsen JB Andersen HR Grand jean P. Plasma malondialdehyde as a biomarker for oxidative stress: Reference interval and effects of life style factors. Clin Chem 1997; 43: 1209-14.

14. Kalra J Chaudhary AK Prasad K. Increased production of oxygen free radicals in cigarette smokers. Int J Exp Pathol 1991; 72: 1-7.

15. Shah AA. Evaluation of Xanthine oxidase Malondialdehyde Ascorbic Acid and Alpha Tocopherol levels in the blood samples of smokers and non smokers. [ Thesis] Pakistan: Isra University.2010.

16. Peluffo G Calcerrada P Piacenza LPizzano N and Radi R. Superoxide-mediated inactivation of nitric oxide and peroxynitrite formation by tobacco smoke in vascular epithelium: studies in cultured cells and smokers. Am J Physiol Heart Circ Physiol 2009; 296: H1781-92.
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Publication:Journal of Pakistan Medical Association
Date:Jan 31, 2015
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